The establishment of proper cell polarity is crucial for the structure and function of many tissues, and the loss of polarity is a hallmark of cancer cells. Several known polarity proteins are tumor suppressors, and down regulation of apical polarity proteins induces epithelial-to-mesencyhmal transition. The mechanisms by which polarity proteins are initially localized during de novo assembly of epithelial tissues are largely unknown. To understand polarity establishment and maintenance, we need to understand the interactions between the network of polarity proteins, their functions, and how the interactions are regulated. The cellularizing Drosophila embryo provides a unique system in which to study the establishment of epithelial cell polarity. My goal is to determine how polarity is established and maintained, by defining the hierarchy of polarity proteins, their interactions with each other, and their functions. In the current model, Par-3 (Bazooka in flies) is the most upstream protein in the polarity hierarchy. Its apical localization directs the apical localization of adherens junction an apical proteins. Recently, the Peifer lab found that the afadin homolog Canoe and the GTPase Rap1 are required for proper apical localization of Bazooka. The mechanism by which they do this is unknown. I will generate cno mutants to define how Canoe localizes Bazooka to the apical domain. Furthermore, I will use FRET on live embryos to determine if Rap1 activity is spatially and temporally regulated, and I will define which of Rap1 GEFs and downstream effectors regulate cell polarity. Finally, I will identify basolateral proteins that are important n establishing or maintaining apical polarity. I will define their mechanism of action by examining trafficking of apical proteins in each line and looking for alterations in protein dynamics. How tissues polarize is clearly more complex than originally hypothesized. Though many polarity proteins have been identified, the functions of many during the initiation of polarity have not been addressed, and the interactions within the network of polarity proteins are not well established. Defining the network and elucidating the mechanism of polarity establishment and maintenance is important to understand proper development, but also, to understand where or how the polarity network is disrupted during epithelial-to- mesencyhmal transitions and metastasis of cancer cells.